Compositions and methods for inducing immune tolerance

ABSTRACT

The present invention provides liposomal compositions for inducing immune tolerance. The compounds typically comprise a liposome displaying a specific antigen and also a binding moiety for a sialic acid binding Ig-like lectin (Siglec)expressed on B cells. The invention also provides methods for inducing tolerance to a protein or polypeptide antigen (e.g., a protein antigen) in a subject. The methods involve administering to the subject a pharmaceutical composition that co-presents both the antigen and a glycan ligand for a Siglec expressed on B lymphocytes.

CROSS-REFERENCE TO RELATED APPLICATIONS

The subject patent application claims the benefit of priority to U.S.Provisional Patent Application Nos. 61/400,610 (filed Jul. 31, 2010) and61/464,136 (filed Feb. 28, 2011). The full disclosures of the priorityapplications are incorporated herein by reference in their entirety andfor all purposes.

STATEMENT OF GOVERNMENT SUPPORT

This invention was made with government support under Contract Nos.GM060938, AI050143, and GM044809 awarded by the National Institutes ofHealth. The government has certain rights in this invention.

BACKGROUND OF THE INVENTION

Immune responses are necessary for protection against potentiallypathogenic microorganisms. However, undesired immune activation cancause injurious processes leading to damage or destruction of one's owntissues. Undesired immune activation occurs, for example, in autoimmunediseases where antibodies and/or T lymphocytes react with self antigensto the detriment of the body's tissues. This is also the case inallergic reactions characterized by an exaggerated immune response tocertain environmental matters and which may result in inflammatoryresponses leading to tissue destruction. This is also the case inrejection of transplanted organs which is significantly mediated byalloreactive T cells present in the host which recognize donoralloantigens or xenoantigens.

Immune tolerance is the acquired lack of specific immune responsivenessto an antigen to which an immune response would normally occur.Typically, to induce tolerance, there must be an exposure to atolerizing antigen, which results in the death or functionalinactivation of certain lymphocytes. This process generally accounts fortolerance to self antigens, or self-tolerance. Immunosuppressive agentsare useful in prevention or reduction of undesired immune responses,e.g., in treating patients with autoimmune diseases or with allogeneictransplants. However, immunosuppressive agents can also cause systemicimmune suppression, toxicity and even death due to opportunisticinfections.

There is a need in the art for safer and more effective means forinducing immune tolerance, especially antigen-specific immune tolerance.The instant invention addresses this and other needs.

SUMMARY OF THE INVENTION

In one aspect, the invention provides liposomal compositions forinducing immune tolerance to specific antigens. The compositionstypically contain a liposome nanoparticle which displays on its surfaceboth a specific antigen and also a ligand or a binding moiety for asialic acid binding Ig-like lectin (Siglec). In some preferredembodiments, the ligand or binding moiety is a glycan ligand for theSiglec. Some of the liposomal compositions contain a ligand or bindingmoiety for a Siglec expressed on B lymphocytes, e.g., Siglec-2 (CD22) orSiglec-G/10.

In some embodiments of the invention, the liposomal compositions areintended for targeting a Siglec expressed on human lymphocytes. In someof these embodiments, the employed Siglec glycan ligand is9-N-biphenylcarboxyl-NeuAcα2-6Galβ1-4GlcNAc (6′-BPCNeuAc),NeuAcα2-6Galβ1-4GlcNAc or NeuAcα2-6Galβ1-4(6-sulfo)GlcNAc. The specificantigen against which immune tolerance is to be induced can be aprotein. The antigen can also be a hapten, a carbohydrate, or a nucleicacid. In related aspects, pharmaceutical compositions and kitscomprising the liposomal composition of the invention are also provided.

In a related aspect, the invention provides methods for inducingtolerance to a T-dependent antigen in a subject (e.g. a specificpolypeptide or protein antigen). These methods entail administering tothe subject a pharmaceutical composition that contains an effectiveamount of the antigen and a ligand for a Siglec expressed on Blymphocytes. In some embodiments, the antigen is conjugated to theSiglec ligand covalently or non-covalently. In some other embodiments,the antigen and the Siglec ligand are co-displayed on a liposome.

Some embodiments are directed to inducing immune tolerance to anautoantigen, an allergen, or an alloantigen. In some embodiments, theemployed Siglec ligand is a glycan ligand that specifically recognizes aSiglec expressed on B lymphocytes, e.g., CD22 or Siglec-G/10. Somepreferred embodiments of the invention are directed to inducing immunetolerance in a human subject. In these embodiments, a Siglec ligand thatspecifically recognizes a human Siglec is used, e.g.,9-N-biphenylcarboxyl-NeuAcα2-6Galβ1-4GlcNAc (6′-BPCNeuAc),NeuAcα2-6Galβ1-4GlcNAc, or NeuAcα2-6Galβ1-4(6-sulfo)GlcNAc.

In another aspect, the invention provides methods for suppressing orpreventing an undesired immune response to a specific antigen in asubject. The methods involve administering to the subject apharmaceutical composition containing a liposome composition thatdisplays both the antigen and a ligand for a sialic acid binding Ig-likelectin (Siglec). The Siglec can be one expressed on the surface of Blymphocytes or one expressed on the surface of other leukocytes such asmonocytes or macrophages. In some embodiments, the antigen displayed onthe liposomal composition is an autoantigen, an allergen, or analloantigen. Some embodiments employ a glycan ligand that specificallyrecognizes a Siglec expressed on B cells, e.g., CD22 or Siglec-G/10. Insome preferred embodiments, the subject to be treated is a human. Someof these embodiments employ a Siglec glycan ligand that recognizes humanCD22 or Siglec-10, e.g., 9-N-biphenylcarboxyl-NeuAcα2-6Galβ1-4GlcNAc(6′-BPCNeuAc) or 9-N-biphenylcarboxyl-NeuAcα2-3Galβ1-4GlcNAc(3′-BPCNeuAc).

A further understanding of the nature and advantages of the presentinvention may be realized by reference to the remaining portions of thespecification and claims.

DESCRIPTION OF THE DRAWINGS

FIG. 1 shows that liposomes displaying a synthetic glycan ligand ofmouse CD 22, ^(BPA)NeuGc, and hen egg lysozyme (HEL) induce tolerance toHEL in mice.

FIG. 2 shows that liposomes displaying HEL and a low affinity naturalligand of mouse CD22 can also induce tolerance to HEL in mice.

FIGS. 3A and 3B show that no tolerization to HEL is achieved when usingliposomes co-presenting HEL and (A) ^(BPA)NeuGc or (B) NeuGc in CD22knockout mice.

FIG. 4 shows that co-presentation of ^(BPA)NeuGc and HEL on liposomesgreatly reduces the proliferation of HEL-specific B cells in vitro.

FIG. 5 shows that co-presentation of ^(BPA)NeuGc and HEL on liposomescauses apoptosis of HEL-specific B cells in vitro.

FIG. 6 shows that co-presentation of ^(BPA)NeuGc and HEL on liposomesgreatly reduces the proliferation of HEL-specific B cells in vivo.

FIG. 7 shows that liposomes displaying BPANeuGc and nitrophenyl (NP)induces tolerance to NP in mice.

DETAILED DESCRIPTION

I. Overview

The present invention is predicated in part on the present inventors'discoveries that co-presentation of a T-dependent (protein) antigen withSiglec ligands can induce tolerance to the antigen. In addition, it wasdemonstrated that a liposomal formulation displaying an antigen and aSiglec ligand can induce tolerance to either a T-dependent (protein) orT-independent (hapten) antigen. Specifically, profound systemic B celltolerance to an antigen (e.g., a T-dependent protein antigen or aT-independent small molecule hapten) was induced in animals byadministration of liposomal nanoparticles that displays both the antigenand high affinity ligands of B cell Siglecs (CD22 and Siglec-G).Subsequent administration of antigen 15 or 30 days later results in aseverely blunted production of antibody. It was also shown that thepresence of the Siglec ligand on the antigen bearing liposome not onlysuppresses B cell activation, but also induces apoptosis of the cellthat recognizes the antigen.

The present invention accordingly provides methods and compositions forsuppressing undesired immune responses (e.g., autoimmune responses) andinducing systemic immune tolerance to any specific antigen. Someembodiments of the invention relate to suppressing immune responses andinducing tolerance to protein or polypeptide antigens by co-presenting aT cell-dependent antigen and a high affinity Siglec ligand. Some otherembodiments relate to suppressing immune response and inducing toleranceto any specific antigen via a liposomal composition displaying both theantigen and a high affinity Siglec ligand. The invention also providesliposomal compositions bearing (or immune-conjugates harboring) a Siglecligand and a specific antigen for inducing systemic tolerance to theantigen in a subject. The following sections provide more detailedguidance for practicing the invention.

II. Definitions

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by those of ordinary skillin the art to which this invention pertains. The following referencesprovide one of skill with a general definition of many of the terms usedin this invention: Academic Press Dictionary of Science and Technology,Morris (Ed.), Academic Press (1^(st) ed., 1992); Oxford Dictionary ofBiochemistry and Molecular Biology, Smith et al. (Eds.), OxfordUniversity Press (revised ed., 2000); Encyclopaedic Dictionary ofChemistry, Kumar (Ed.), Anmol Publications Pvt. Ltd. (2002); Dictionaryof Microbiology and Molecular Biology, Singleton et al. (Eds.), JohnWiley & Sons (3^(rd) ed., 2002); Dictionary of Chemistry, Hunt (Ed.),Routledge (1^(st) ed., 1999); Dictionary of Pharmaceutical Medicine,Nahler (Ed.), Springer-Verlag Telos (1994); Dictionary of OrganicChemistry, Kumar and Anandand (Eds.), Anmol Publications Pvt. Ltd.(2002); and A Dictionary of Biology (Oxford Paperback Reference), Martinand Hine (Eds.), Oxford University Press (4^(th) ed., 2000). Furtherclarifications of some of these terms as they apply specifically to thisinvention are provided herein.

The term “agent” includes any substance, molecule, element, compound,entity, or a combination thereof. It includes, but is not limited to,e.g., protein, polypeptide, small organic molecule, polysaccharide,polynucleotide, and the like. It can be a natural product, a syntheticcompound, or a chemical compound, or a combination of two or moresubstances. Unless otherwise specified, the terms “agent”, “substance”,and “compound” are used interchangeably herein.

The term “analog” or “derivative” is used herein to refer to a moleculethat structurally resembles a reference molecule (e.g., a known Siglecligand) but which has been modified in a targeted and controlled manner,by replacing a specific substituent of the reference molecule with analternate substituent. Compared to the reference molecule, an analogwould be expected, by one skilled in the art, to exhibit the same,similar, or improved utility. Synthesis and screening of analogs toidentify variants of known compounds having improved traits (such ashigher binding affinity for a target molecule) is an approach that iswell known in pharmaceutical chemistry.

The term antigen broadly refers to a molecule that can be recognized bythe immune system. It encompasses proteins, polypeptides,polysaccharides, small molecule haptens, nucleic acids, as well aslipid-linked antigens (polypeptide- or polysaccharide-linked lipids.

T cell-dependent or T-dependent antigens refer to antigens which requireT cell assistance in eliciting antibody production by B cells.Structurally these antigens are characterized by multiple antigenicdeterminants. Proteins or polypeptides are typical examples ofT-dependent antigens that contain antigenic determinants for both B andT cells. With a T-dependent antigen, the first signal comes from antigencross linking of the B cell receptor (BCR) and the second signal comesfrom co-stimulation provided by a T cell. T-dependent antigens containantigenic peptides that stimulate the T cell. Upon ligation of the BCR,the B cell processes the antigen, releasing antigenic peptides that arepresented on B cell Class II MHC to a special subtype of T cell called aTh2 cell. The Th2 cell then secretes potent cytokines that activate theB cell. These cytokines trigger B cell proliferation, induce the B cellsto produce antibodies of different classes and with increased affinity,and ultimately differentiate into antibody producing plasma cells.

T cell-independent or T-independent (TI) antigens are antigens which candirectly stimulate the B cells to elicit an antibody response, do notcontain proteins, and cannot induce T cell help. Typically,T-independent antigens have polymeric structures, e.g., the sameantigenic determinant repeated many times. Examples of T-independentantigens include small molecule haptens, nucleic acids, carbohydratesand polysaccharides.

As used herein, immune tolerance (or simply “tolerance”) is the processby which the immune system does not attack an antigen. It occurs inthree forms: central tolerance, peripheral tolerance and acquiredtolerance. Tolerance can be either “natural” or “self tolerance”, wherethe body does not mount an immune response to self antigens, or “inducedtolerance”, where tolerance to antigens can be created by manipulatingthe immune system. When tolerance is induced, the body cannot produce animmune response to the antigen. Mechanisms of tolerance and toleranceinduction are complex and poorly understood. As is well known in the art(see, e.g., Basten et al., Curr. Opinion Immunol. 22:566-574, 2010),known variables in the generation of tolerance include thedifferentiation stage of the B cell when antigen is presented, the typeof antigen, and the involvement of T cells and other leukocytes inproduction of cytokines and cofactors. Thus, suppression of B cellactivation cannot be equated with immune tolerance. For example, while Bcell activation can be inhibited by cross-linking CD22 to the BCR, theselective silencing of B cells does not indicate induction of tolerance.See, e.g., Nikolova et al., Autoimmunity Rev. 9:775-779, 2010; Mihaylovaet al., Mol. Immunol. 47:123-130, 2009; and Courtney et al., Proc. Natl.Acad. Sci. 106:2500-2505, 2009.

The term “immune conjugate” as used herein refers to a complex in whicha Siglec ligand is coupled to an antigen. The Siglec ligand can becoupled directly to the antigen via an appropriate linking chemistry.Alternatively, the Siglec ligand is linked indirectly to the antigen,e.g., via a third molecule such as a spacer or a lipid moiety on aliposome. The linkage between the antigen and the Siglec ligand can beeither covalent or non-covalent.

A “liposomal composition” as used herein refers to a complex thatcontains a lipid component that forms a bilayer liposome structure. Itis typically a semi-solid, ultra fine vesicle sized between about 10 andabout 200 nanometers. The liposomal composition displays on orincorporates into the lipid moiety a binding moiety (e.g., a glycanligand) that is specific for a target molecule (e.g., a Siglec) on atarget cell. Typically, the binding moiety is integrated into the lipidcomponent of the liposome complex. The liposomal compositionadditionally also displays a biological agent (e.g., an antigen) that isto be delivered to a target cell. The biological agent is typically alsointegrated into the lipid component of the liposome complex. Unlessotherwise noted, the biological agent (e.g., an antigen) is not presentin an aqueous solution encapsulated inside the lipid bilayer of theliposome.

Siglecs, short for sialic acid binding Ig-like lectins, are cell surfacereceptors and members of the immunoglobulin superfamily (IgSF) thatrecognize sugars. Their ability to recognize carbohydrates using animmunoglobulin domain places them in the group of I-type (Ig-type)lectins. They are transmembrane proteins that contain an N-terminalV-like immunoglobulin (IgV) domain that binds sialic acid and a variablenumber of C2-type Ig (IgC2) domains. The first described Siglec issialoadhesin (Siglec-1/CD169) that is a lectin-like adhesion molecule onmacrophages. Other Siglecs were later added to this family, includingCD22 (Siglec-2) and Siglec-G/10 (i.e., human Siglec-10 and mouseSiglec-G), which is expressed on B cells and has an important role inregulating their adhesion and activation, CD33 (Siglec-3) andmyelin-associated glycoprotein (MAG/Siglec-4). Several additionalSiglecs (Siglecs 5-12) have been identified in humans that are highlysimilar in structure to CD33 so are collectively referred to as‘CD33-related Siglecs’. These Siglecs are expressed on human NK cells, Bcells, and/or monocytes. CD33-related Siglecs all have two conservedimmunoreceptor tyrosine-based inhibitory motif (ITIM)-like motifs intheir cytoplasmic tails suggesting their involvement in cellularactivation. Detailed description of Siglecs is provided in theliterature, e.g., Crocker et al., Nat. Rev. Immunol. 7:255-66, 2007;Crocker et al., Immunol. 103:137-45, 2001; Angata et al., Mol. Diversity10:555-566, 2006; and Hoffman et al., Nat. Immunol. 8:695-704, 2007.

Glycan ligands of Siglecs refer to compounds which specificallyrecognize one or more Siglecs and which comprise homo- or heteropolymersof monosaccharide residues. In addition to glycan sequences, the Siglecglycan ligands can also contain pegylated lipid moiety connected to theglycan via a linker. Examples of various Siglec glycan ligands arereported in the literature, e.g., Paulson et al., WO 2007/056525; andBlixt et al., J. Am. Chem. Soc. 130:6680-1, 2008.

Leukocytes refer to cells of the immune system which are involved indefending the body against both infectious disease and foreignmaterials. More specifically, the term as used in the invention refersto mononuclear leukocytes including lymphocytes, monocytes, andmacrophages.

Administration “in conjunction with” one or more other therapeuticagents includes simultaneous (concurrent) and consecutive administrationin any order.

The term “contacting” has its normal meaning and refers to combining twoor more agents (e.g., polypeptides or small molecule compounds) orcombining agents with cells. Contacting can occur in vitro, e.g.,combining an agent with a cell or combining two cells in a test tube orother container. Contacting can also occur in vivo, e.g., by targeteddelivery of an agent to a cell inside the body of a subject.

The term “subject” refers to any animal classified as a mammal, e.g.,human and non-human mammals. Examples of non-human animals include dogs,cats, cattle, horses, sheep, pigs, goats, rabbits, and etc. Unlessotherwise noted, the terms “patient” or “subject” are used hereininterchangeably. Preferably, the subject is human.

The term “treating” or “alleviating” includes the administration ofcompounds or agents to a subject to prevent or delay the onset of thesymptoms, complications, or biochemical indicia of a disease (e.g., anautoimmune disease), alleviating the symptoms or arresting or inhibitingfurther development of the disease, condition, or disorder. Subjects inneed of treatment include those already suffering from the disease ordisorder as well as those being at risk of developing the disorder.Treatment may be prophylactic (to prevent or delay the onset of thedisease, or to prevent the manifestation of clinical or subclinicalsymptoms thereof) or therapeutic suppression or alleviation of symptomsafter the manifestation of the disease.

III. Antigens for Inducing Immune Tolerance

The invention provides compositions and methods for inducing systemictolerance to a specific antigen. Various antigens can be used inpreparing liposomal compositions or immune-conjugates for inducingimmune tolerance as disclosed herein. The antigens include various Tcell-dependent antigens or T cell-independent antigens. In someembodiments, the employed antigen is one that is involved in anundesired immune reaction or response. In some embodiments, the employedantigen is any antigen to which an individual may be at risk ofdeveloping an undesired immune reaction or response. For example, someembodiments of the invention are directed to compositions and methodsfor inducing immune tolerance against autoantigens, alloantigens orallergens.

Antigens of different chemical nature are suitable for inducing immunetolerance with methods of the invention. They include polypeptides orproteins, haptens, carbohydrates, nucleic acids, peptides, polyethyleneglycol, lipids (e.g., sterols excluding cholesterol, fatty acids, andphospholipids), polysaccharides, and gangliosides. The various antigenssuitable for practicing the present invention may be isolated from theirsource using purification techniques known in the art or, moreconveniently, may be produced using recombinant methods. For example,the antigens can be obtained through a number of methods known in theart, including isolation and synthesis using chemical and enzymaticmethods. In certain cases, such as for many sterols, fatty acids andphospholipids, the antigenic portions of the molecules are commerciallyavailable. Antigens derived from infectious agents may be obtained usingmethods known in the art, for example, from native viral or bacterialextracts, from cells infected with the infectious agent, from purifiedpolypeptides, from recombinantly produced polypeptides and/or assynthetic peptides.

Some embodiments of the invention are intended for inducing immunetolerance to various autoantigens. Autoantigens are known for a numberof autoimmune diseases. For example, Grave's disease is characterized byproduction of autoantibodies to the thyroid-stimulating hormone receptorof the thyroid gland, Hashimoto's thyroiditis by autoantibodies and Tcells to thyroid antigens (e.g., thyroid peroxidase), and type Idiabetes by T cells and autoantibodies to β cell antigens (e.g.,glutamic acid decarboxylase and insulin). Other examples of autoantigensinvolved in autoimmune diseases include, but are, not limited to,cytochrome P450 antigens in Addison's disease, myelin proteins (e.g.,myelin basic protein) in MS, uveal antigens in uveitis, gastric parietalcell antigens (e.g., H⁺/ATPase, intrinsic factor) in pernicious anemia,transglutaminase in gluten enteropathy, myocardial cell proteins (e.g.,myosin) in myocarditis and rheumatic heart disease, platelet antigens(e.g., GP IIb/IIIa) in idiopathic thrombocytopenic purpura, red bloodcell membrane proteins in autoimmune hemolytic anemia, neutrophilmembrane proteins in autoimmune neutropenia, basement membrane antigens(e.g., type IV collagen .alpha.3 chain) in Goodpasture's disease,intrahepatic bile duct/mitochondrial antigens (e.g., 2-oxoaciddehydrogenase complexes) for primary biliary cirrhosis, hepatocyteantigens (e.g., cytochrome P450, 206) for autoimmune hepatitis,acetylcholine receptors for myasthenia gravis, and desmogleins forpemphigus and other bullous diseases.

Some embodiments of the invention are directed to inducing toleranceagainst protein antigens that are normally self antigens, but whichcertain individuals lack owing to genetic deficiency and to whichunwanted immune reactions occur upon replacement therapy. Examples ofsuch antigens include blood coagulation factors VIII and IX in subjectswith hemophilia A and B (see, e.g., van Helden et al., Haemophilia.16:35-43, 2010; and DiMichele, Br J Haematol. 138:305-15, 2007),alpha-L-iduronidase in subjects with Hurler syndrome (see, e.g.,Kakavanos et al., FEBS Lett. 580:87-92, 2006), and adenosine deaminasein subjects with adult-type adenosine deaminase deficiency (see, e.g.,Bax et al., Eur. J. Haematol. 79:338-48, 2007).

Some embodiments of the invention are directed to inducing immunetolerance to allergens. Any allergen can be employed in the practice ofthe invention. For example, various allergens from food are suitable forpractice of the invention. Example of such allergens include peanutallergen (e.g., Ara h I or Ara h II); walnut allergen (e.g., Jug r I);brazil nut allergen (e.g., albumin); shrimp allergen (e.g., Pen a I);egg allergen (e.g., ovomucoid); milk allergen (e.g., bovineβ-lactoglobin); wheat gluten antigen (e.g., gliadin); and fish allergen(e.g., parvalbumins). In some embodiments, the employed allergen is alatex allergen such as Hey b 7 (Sowka et al., Eur. J. Biochem.255:213-219, 1998). In addition to food allergens, other types ofallergens can also be used in the practice of the invention. Examples ofsuch allergens including, but not limited to, ragweed pollen allergenAntigen E (Amb a I) (Rafnar et al., J. Biol. Chem. 266:1229-1236, 1991),grass allergen Lol p 1 (Tamborini et al., Eur. J. Biochem. 249:886-894,1997), major dust mite allergens Der pI and Der PII (Chua et al., J.Exp. Med 167:175-182, 1988; Chua et al., Int. Arch. Allergy Appl.Immunol. 91:124-129, 1990), domestic cat allergen Fel d I (Rogers etal., Mol. Immunol. 30:559-568, 1993), white birch pollen Bet vl(Breiteneder et al., EMBO J. 8:1935-1938, 1989), Japanese cedarallergens Cry j 1 and Cry j 2 (Kingetsu et al., Immunol. 99:625-629,2000), and protein antigens from other tree pollen (Elsayed et al.,Scand. J. Clin. Lab. Invest. Suppl. 204:17-31, 1991). Also suitable forthe invention are protein antigens from grass pollen and known allergensfrom trees, including allergens from birch, juniper and Japanese cedar.

In some embodiments, the employed antigen for inducing immune toleranceis an alloantigen. Alloantigens are generally cellular antigens thatvary in structure among individual members of a single species.Alloantigens from one individual can be recognized as foreign antigensby other members of the same species and are often the basis for graftrejection reactions. Examples of alloantigens include, but are notlimited to major histocompatability complex (MHC) class I and class IIantigens, minor histocompatability antigens, certain tissue-specificantigens, endothelial glycoproteins such as blood group antigens, andcarbohydrate determinants.

IV. Siglec Ligands and Complexes with Antigens for Inducing Tolerance

The present invention provides immune-conjugates which contain a bindingmoiety (e.g., a Siglec ligand) that is directly or indirectly linked toa specific antigen. Some of the immune-conjugates are specificallydesigned for inducing immune tolerance to protein antigens or otherT-dependent antigens. The invention also provides liposomal compositions(or liposome complexes) that are suitable for inducing immune toleranceor suppressing immune response to a specific antigen. The liposomalcompositions typically display both the specific antigen and a bindingmoiety that specifically recognizes a Siglec on a target cell (e.g., Blymphocytes or monocytes). The specific antigen displayed on theliposomal compositions can be any antigen described herein, includingany T-dependent or T-independent antigen known in the art. In addition,the binding moiety of the liposomal compositions or immune conjugates ofthe invention typically contains a ligand for the Siglec.

The Siglec ligands suitable for the invention include ligands forvarious Siglec molecules. Some preferred embodiments of the inventionemploy glycan ligands directed again Siglecs that are expressed on thesurface of B lymphocytes. For example, the ligands can be natural orsynthetic ligands that specifically recognize CD22 (Siglec-2) and/orSiglec G/10. CD22 from a number of species are known in the art. Forexample, amino acid sequences for human CD22 are disclosed in theNational Center for Biotechnology Information (NCBI) database(http://www.ncbi.nlm.nih.gov/) at accession number NP 001762 (gi:4502651) and also available in WO 2007/056525. Mouse CD22 is alsocharacterized in the art, e.g., Torres et al., J. Immunol. 149:2641-9,1992; and Law et al., J Immunol. 155:3368-76, 1995. Other than CD22,Siglec-G/10 is another Siglec expressed on the surface of B cells. HumanSiglec-10 and its mouse ortholog Siglec-G are both well known andcharacterized in the art. See, e.g., Munday et al., Biochem. J.355:489-497, 2001; Whitney et al., Eur. J. Biochem. 268:6083-96, 2001;Hoffman et al., Nat. Immunol. 8:695-704, 2007; and Liu et al., TrendsImmunol. 30:557-61, 2009.

Various ligands of CD22 and Siglec-G/10 are known and suitable for thepractice of the present invention. See, e.g., Paulson et al., WO2007/056525; Chen et al., Blood 115:4778-86, 2010; Blixt et al., J. Am.Chem. Soc. 130:6680-1,2008; Kumari et al., Virol. J. 4:42, 2007; andKimura et al., J. Biol. Chem. 282:32200-7, 2007. For example, naturalligands of human CD22 such as NeuAcα2-6Galβ1-4GlcNAc, orNeuAcα2-6Galβ1-4(6-sulfo)GlcNAc can be used for targeting an antigen tohuman B cells. In addition, a number of synthetic CD22 ligands withimproved activities are also available, e.g.,9-N-biphenylcarboxyl-NeuAcα2-6Galβ1-4GlcNAc (6′-BPCNeuAc) and9-N-biphenylcarboxyl-NeuAcα2-3Ga1β1-4GlcNAc (3′-BPCNeuAc). More specificglycan ligands for human CD22 or Siglec-10 are described in the art,e.g., Blixt et al., J. Am. Chem. Soc. 130:6680-1, 2008; and Paulson etal., WO 2007/056525. Similarly, many glycan ligands for mouse CD22 havebeen reported in the literature. Examples include NeuGcα2-6Galβ1-4GlcNAc(NeuGc), 9-N-biphenylacetyl-NeuGcα2-6Galβ1-4GlcNAc (^(BPA)NeuGc), andNeuGcα2-3Galβ1-4GlcNAc. Some of these CD22 ligands are also known to beable to bind to Siglec-G/10. Other than the natural and synthetic Siglecligands exemplified herein, one can also employ derivative or analogcompounds of any of these exemplified glycan ligands in the practice ofthe invention.

Some embodiments of the present invention relate to liposomalcompositions (or liposome targeting compositions or complexes) forinducing systemic immune tolerance to a specific antigen. Typically, theliposomal compositions display on the surface of a liposome both aspecific antigen and a binding moiety that specifically recognizes aSiglec on a target cell (e.g., B cell or monocytes). The binding moietyis a molecule that recognizes, binds or adheres to a target moleculelocated in a cell, tissue (e.g. extracellular matrix), fluid, organism,or subset thereof. The binding moiety and its target molecule representa binding pair of molecules, which interact with each other through anyof a variety of molecular forces including, for example, ionic,covalent, hydrophobic, van der Waals, and hydrogen bonding, so that thepair have the property of binding specifically to each other. Specificbinding means that the binding pair exhibit binding with each otherunder conditions where they do not bind to another molecule. In somepreferred embodiments, the binding moiety present on the liposomalcomposition is a glycan ligand that specifically recognizes a Siglec(e.g., CD22 or Siglec-G/10) expressed on the surface of B cells. Inaddition to the binding moiety, the liposome compositions of theinvention also bear or display a specific antigen against which immunetolerance is to be induced. Any antigens (including but not limited toT-dependent antigen or T-independent antigens) described herein or wellknown in the art can be employed in preparing the liposomal composition.

The liposome component of the compounds of the invention is typically avesicular structure of a water soluble particle obtained by aggregatingamphipathic molecules including a hydrophilic region and a hydrophobicregion. While the liposome component is a closed micelle formed by anyamphipathic molecules, it preferably includes lipids. For example, theliposomes of the invention exemplified herein contain phospholipids suchas distearoyl phosphatidylcholine (DSPC) andpolyethyleneglycol-distearoyl phosphoethanolamine (PEG-DSPE). Otherphospholipids can also be used in preparing the liposomes of theinvention, including dipalmitoylphosphatidylcholine (DPPC),dioleylphosphatidylcholine (DOPC) and dioleylphosphatidyl ethanolamine(DOPE), sphingoglycolipid and glyceroglycolipid. These phospholipids areused for making the liposome, alone or in combination of two or more orin combination with a lipid derivative where a non-polar substance suchas cholesterol or a water soluble polymer such as polyethylene glycolhas been bound to the lipid.

The liposomal compositions of the invention can be prepared inaccordance with methods well known in the art. For example,incorporation of a Siglec ligand and an antigen on the surface of aliposome can be achieved by any of the routinely practiced procedures.Detailed procedures for producing a liposome nanoparticle bearing abinding moiety and an antigen are also exemplified in the Examplesherein. These include liposomes bearing an incorporated glycan ligand(e.g., ^(BPA)NeuGc) and also a specific antigen (e.g., nitrophenyl orhen egg lysozyme). In addition to the methods and procedures exemplifiedherein, various methods routinely used by the skilled artisans forpreparing liposomes can also be employed in the present invention. Forexample, the methods described in Chen et al., Blood 115:4778-86, 2010;and Liposome Technology, vol. 1, 2^(nd) edition (by Gregory Gregoriadis(CRC Press, Boca Raton, Ann Arbor, London, Tokyo), Chapter 4, pp 67-80,Chapter 10, pp 167-184 and Chapter 17, pp 261-276 (1993)) can be used.More specifically, suitable methods include, but are not limited to, asonication method, an ethanol injection method, a French press method,an ether injection method, a cholic acid method, a calcium fusionmethod, a lyophilization method and a reverse phase evaporation method.A size of the liposome of the present invention is not particularlylimited, and typically is preferably between 1 to 200 nm and morepreferably between 10 to 100 nm in average. The structure of theliposome is not particularly limited, and may be any liposome such asunilamella and multilamella. As a solution encapsulated inside theliposome, it is possible to use buffer and saline and others in additionto water.

Other than the liposomal compositions described above, some otherembodiments of the invention relate to immune conjugates which contain aspecific antigen linked to a ligand for a Siglec expressed on thesurface of a target cell (e.g., B cell or monocytes). Such immuneconjugates can also be readily employed for delivering the antigen tothe target cell and accordingly inducing immune tolerance to theantigen. Some of the immune conjugates are intended to target a proteinor polypeptide antigen via a glycan ligand that recognizes a Siglec(Siglec-2 or Siglec-G/10) expressed on the surface of B cells. Anyprotein antigen or T-dependent antigen can be used for preparing suchimmune conjugates. Suitable ligands for targeting the antigen to B cellsare also described herein. Conjugating a protein or polypeptide to asmall binding ligand can be performed in accordance with methods wellknown in the art. See, e.g., Chemistry of protein conjugation andcross-linking, Shan Wong, CRC Press (Boca Raton, Fla., 1991); andBioconjugate techniques, 2^(nd) ed., Greg T. Hermanson, Academic Press(London, UK, 2008).

V. Inducing Immune Tolerance via Siglec Ligand-Mediated AntigenTargeting

The invention provides methods and therapeutic uses for suppressingundesired immune responses and/or inducing immune tolerance to aspecific antigen. The compositions described herein can be used fortreating or preventing various diseases or disorders which areassociated with or mediated by an undesired immune response or immuneactivation. By targeting a specific antigen to B cells and/or otherleukocytes (e.g., monocytes and macrophage) in a subject in need oftreatment, the liposomal compositions or immune-conjugates of theinvention are suitable for treating or ameliorating the symptoms ofautoimmune diseases, allergies, asthma, graft-versus-host reactions orgraft rejection reaction.

Some embodiments of the invention are directed to inducing immunetolerance in a subject by using the liposomal compositions disclosedherein to target a specific antigen to B cells in a subject. The antigencan be any antigen against which an immune tolerance is desired. Theseinclude both T-dependent antigens (e.g., protein or polypeptideantigens) and T-independent antigens (e.g., haptens or polysaccharides).In some other embodiments of the invention, immune tolerance against aprotein or polypeptide antigen is induced by targeting to B cells animmune conjugate described herein which contains both a Siglec ligandand the specific antigen. In various embodiments, the liposomecompositions or immune conjugates can be used for delivering an antigento B cells either in vitro or in vivo. Preferably, the liposomalcomposition or immune conjugate bearing both the Siglec ligand and thespecific antigen is administered to a subject in vivo. By inducingimmune tolerance and suppressing undesired immune response, the methodsand compositions described herein find uses in the treatment of variousdiseases and disorders. In any of these applications, the liposomecompositions or immune conjugates disclosed herein can be used alone oradministered in conjunction with other known drugs in the treatment of aspecific disease or condition. The invention further provides for apharmaceutical combination (e.g., a kit) for carrying out thesetherapeutic applications. Such pharmaceutical combination can contain aliposomal composition or immune conjugate disclosed herein, in free formor in a composition, an optional co-agent or carrier, as well asinstructions for administration of the agents.

The liposomal compositions or immune conjugates described herein can beadministered alone or as a component of pharmaceutical compositions.Pharmaceutical compositions of the invention comprise an effectiveamount of the liposomal compositions or immune conjugates formulatedwith at least one pharmaceutically acceptable carrier. Pharmaceuticalcompositions of the invention can be prepared and administered to asubject by any methods well known in the art of pharmacy. See, e.g.,Goodman & Gilman's The Pharmacological Bases of Therapeutics, Hardman etal., eds., McGraw-Hill Professional (10^(th) ed., 2001); Remington: TheScience and Practice of Pharmacy, Gennaro, ed., Lippincott Williams &Wilkins (20^(th) ed., 2003); and Pharmaceutical Dosage Forms and DrugDelivery Systems, Ansel et al. (eds.), Lippincott Williams & Wilkins(7^(th) ed., 1999). In addition, the pharmaceutical compositions of theinvention may also be formulated to include other medically useful drugsor biological agents.

In some preferred embodiments, the liposomal compositions or immuneconjugates are used for in vivo applications. In these applications, theliposome complexes set forth herein can be administered to a subject inneed of treatment according to protocols already well established in theart. The liposomal compositions or immune conjugates can be administeredalone or in combination with a carrier in an appropriate pharmaceuticalcomposition. Typically, a therapeutically effective amount of theliposomal compositions or immune conjugates is combined with apharmaceutically acceptable carrier. The pharmaceutically acceptablecarrier is any carrier known or established in the art. Exemplarypharmaceutically acceptable carriers include sterile pyrogen-free waterand sterile pyrogen-free saline solution. Other forms ofpharmaceutically acceptable carriers that can be utilized for thepresent invention include binders, disintegrants, surfactants,absorption accelerators, moisture retention agents, absorbers,lubricants, fillers, extenders, moisture imparting agents,preservatives, stabilizers, emulsifiers, solubilizing agents, saltswhich control osmotic pressure, diluting agents such as buffers andexcipients usually used depending on the use form of the formulation.These are optionally selected and used depending on the unit dosage ofthe resulting formulation.

A therapeutically effective amount of the antigen varies depending uponthe disorder that a subject is afflicted with, other known factors ofthe subject such as age, weight, etc., and thus must be determinedempirically in each case. This empirical determination can be made byroutine experimentation. Typically, though, the liposome components maybe used at a ratio of about 200:1 w/w, e.g., 100-300:1 w/w, compared tothe antigen delivered. A typical therapeutic dose of the liposomecomposition is about 5-100 mg per dose, e.g., 10 mg per dose. For anygiven condition or disease, one can prepare a suitable liposomalcomposition which contains an appropriate amount of Siglec ligand and anappropriate antigen in accordance with the present disclosure andknowledge well known in the art , e.g., Springhouse, Physician's DrugHandbook, Lippincott Williams & Wilkins (12^(th) edition, 2007).

For in vivo applications, the liposomal compositions or immuneconjugates can be administered to the patient by any customaryadministration route, e.g., orally, parenterally or by inhalation. Asshown in the Example below, a liposome co-displaying an antigen and aSiglec ligand can be administered to a subject by intravenous injection.In some other embodiments, the liposome complex can be administered to asubject intravascularly. A liposome useful for intravascularadministration can be a small unilamellar liposome, or may be a liposomecomprising PEG-2000. When the composition is parenterally administered,the form of the drug includes injectable agents (liquid agents,suspensions) used for intravenous injection, subcutaneous injection,intraperitoneal injection, intramuscular injection and intraperitonealinjection, liquid agents, suspensions, emulsions and dripping agents.

In some other embodiments, the liposomal composition or immune conjugateis administered orally to a subject. In these embodiments, a form of thedrug includes solid formulations such as tablets, coated tablets,powdered agents, granules, capsules and pills, liquid formulations suchas liquid agents (e.g., eye drops, nose drops), suspension, emulsion andsyrup, inhales such as aerosol agents, atomizers and nebulizers, andliposome inclusion agents. In still some other embodiments, the liposomecomposition is administered by inhalation to the respiratory tract of apatient to target the trachea and/or the lung of a subject. In theseembodiments, a commercially available nebulizer may be used to deliver atherapeutic dose of the liposome complex in the form of an aerosol.

The invention also provides kits useful in therapeutic applications ofthe compositions and methods disclosed herein. Typically, the kits ofthe invention contain one or more liposomal compositions or immuneconjugates described herein. The kits can further comprise a suitableset of instructions, generally written instructions, relating to the useof the compounds for inducing immune tolerance to a specific antigenpresent in the compounds. The liposomal composition or immune conjugatecan be present in the kits in any convenient and appropriate packaging.The instructions in the kits generally contain information as to dosage,dosing schedule, and route of administration for the intended method ofuse. The containers of kits may be unit doses, bulk packages (e.g.,multi-dose packages) or sub-unit doses. Instructions supplied in thekits of the invention are typically written instructions on a label orpackage insert (e.g., a paper sheet included in the kit), butmachine-readable instructions (e.g., instructions carried on a magneticor optical storage disk) are also acceptable.

In some embodiments, kits of the invention comprise materials forproduction of a liposomal composition or immune conjugate comprising aspecific antigen and a Siglec ligand. Generally, these kits containseparate containers of one or more antigens and one or more Siglecligands from which a liposomal composition or immune conjugate can bemade. Additional regents for making the compounds can also be providedin the kits, e.g., reagents for making liposome. The Siglec ligands andthe antigens are preferably supplied in a form which allows formation ofcomplexes upon mixing of the other reagents with the supplied Siglecligand and antigen.

EXAMPLES

The following examples are offered to illustrate, but not to limit thepresent invention. The studies exemplified herein were all performed inaccordance with procedures that have been previously described. See,e.g., Paulson et al., U.S. Patent Application Publication No.20090238837; Duong et al., J. Exp. Med. 207:173, 2010; Chen et al.,Blood 115:4778-86, 2010; and Reulen et al., Bioconjugate Chem.,21:860-866, 2010.

We examined whether liposomes displaying a synthetic glycan ligand ofmouse CD22, ^(BPA)NeuGc, and hen egg lysozyme (HEL) can induce toleranceto HEL in mice. The results are shown in FIG. 1. On day 0, 200 μL ofliposomes (1.25 mM lipid) displaying HEL alone (filled circles) or^(BPA)NeuGc and HEL (open squares) were injected into C57BL/6J mice(n=12 per group) via the lateral tail vein. On various days, the micewere bled and the anti-HEL titer (IgM, IgG₁, and IgG_(2a) isotypes) wasdetermined by an ELISA assay. On days 15 and 30 of the experiment(indicated with the arrows), both groups were challenged with the samequantity of liposomes displaying HEL alone. Shown on the left areaverage values and on the right a scatter plot containing eachindividual data point. Except for day 0, the p value between the twogroups is less than 0.0001.

We then investigated the amount of Siglec ligand in the liposomeformulation that is required to induce tolerance to hen egg lysozyme.Data obtained from the study are shown in FIG. 2. The experiment isessentially the same as described for FIG. 1 except that multipleliposome formulations that differed in the type and amount of Siglecligand were compared. On day 0, 200 μL of liposomes (1.25 mM lipid)displaying HEL (filled circles) and varying amounts of either^(BPA)NeuGc (closed circles) or the natural ligand of CD22,NeuGcα2-6Galβ1-4GlcNAc (closed triangles) were injected into C57BL/6Jmice (n=6 per group) via the lateral tail vein. The ligand concentrationin each preparation is shown as the log of the % of the total molarcontent of lipids, ranging from a 4% to about 0.003%. On days 15 and 30of the experiment, both groups were challenged with the same quantity ofliposomes displaying HEL alone. Shown are the serum titer of antibodiesto HEL on day 44 (IgM, IgG₁, and IgG_(2a) isotypes). Some tolerizationis seen even at the lowest doses of the ^(BPA)NeuGc ligand for IgG₁ andIgG_(2a). Tolerization is also seen with the natural ligand of CD22 atthe highest concentrations, even though it exhibits an affinityapproximately 200 fold lower than the ^(BPA)NeuGc ligand.

To investigate if this tolerization is CD22 dependent, we repeated theanalogous studies with liposomes displaying either 0.3% ^(BPA)NeuGc(FIG. 3A) or 4% NeuGc (FIG. 3B) in CD22 knockout mice. As shown in FIGS.3A and 3B, no tolerance was observed in either case. These resultsclearly demonstrate that these ligands act through CD22.

We next examined whether co-presentation of ^(BPA)NeuGc and HEL onliposomes can reduce the proliferation of HEL-specific B cells in vitro.HEL-specific B cells were purified from the spleen of MD4 mice andfluorescently labeled with carboxyfluorescein succinimidyl ester (CFSE).Liposomes (10 nM final concentration) displaying HEL alone (left panels)or HEL and ^(BPA)NeuGc (right panels) were incubated with 2×10⁵ cells.Some cells were not treated with any liposomes. Using flow cytometry,the dilution of CFSE signal in the cells was monitored on the indicateddays. Cells treated without liposomes are shown in shaded grey in orderto establish the position of the undivided cells. Indicated on eachgraph is the percentage of divided cells and the division index, whichis the average number of cell divisions that cells in the originalpopulation have undergone. The results as shown in FIG. 4 indicate thatco-presentation of ^(BPA)NeuGc and HEL on liposomes greatly reduced theproliferation of HEL-specific B cells in vitro.

We further assessed whether co-presentation of ^(BPA)NeuGc and HEL onliposomes can leads to apoptosis of HEL-specific B cells in vitro.HEL-specific B cells were purified from the spleen of MD4 mice.Liposomes (10 nM final concentration) displaying HEL alone (left panel)or HEL and ^(BPA)NeuGc (right panel) were incubated with 2×10⁵ cells.Some cells were not treated with any liposomes. After two days ofincubation, the extent of apoptosis was determined by staining the cellswith AnnexinV-FITC and analysis by cytometry. For each graph, cellstreated without liposomes are shown in shaded grey. The results from thestudy are shown in FIG. 5. As shown in the figure, co-presentation of^(BPA)NeuGc and HEL on liposomes causes apoptosis of HEL-specific Bcells in vitro.

We additionally studied whether co-presentation of ^(BPA)NeuGc and HELon liposomes greatly reduces the proliferation of HEL-specific B cellsin vivo. In the study, HEL-specific B cells were purified from thespleen of MD4 mice (wild-type or CD22 knockout background) andfluorescently labeled with carboxyfluorescein succinimidyl ester (CFSE).Labeled cells (8×10⁶ or 5×10⁶) were transferred into a host C57BL/6Jmouse via the tail vein. Two hours later, liposomes displaying HEL aloneor HEL and ^(BPA)NeuGc were injected via the tail vein. Several micewere injected with buffer as a control. On the indicated day, the spleenwas removed and dilution of CFSE signal in spleenocytes was determinedby flow cytometry. HEL-specific B cells were gated for by staining withan anti-IgM^(A)-PE antibody. Mice injected with buffer are shown inshaded grey in order to establish the position of the undivided cells.Indicated on each graph is the percentage of divided cells and thedivision index, which is the average number of cell divisions that cellsin the original population have undergone. The results obtain the study,shown in FIG. 6, indicate that co-presentation of ^(BPA)NeuGc and HEL onliposomes also greatly reduces the proliferation of HEL-specific B cellsin vivo. Repeating this experiment with HEL-specific B cells on a CD22deficient background resulted in no difference between the liposomeswith or without BPANeuGc, indicating that the effect is entirelyCD22-dependent.

Finally, we examined whether liposomes displaying ^(BPA)NeuGc andnitrophenyl (NP) can also induce tolerance to NP in mice. Specifically,on day 0, 200 μL of liposomes (2.5 mM lipid) displaying NP alone (filledcircles) or ^(BPA)NeuGc and NP (open squares) were injected intoC57BL/6J mice (n=10 per group) via the lateral tail vein. On variousdays, the mice were bled and the anti-NP titer (IgM and IgG isotypes)was determined by an ELISA assay. On days 14 and 28 of the experiment(indicated with the arrows), both groups were challenged with liposomesdisplaying NP alone. Shown on the left are average values and on theright a scatter plot containing each individual data point. Statisticalsignificance between the two groups at each time point is noted by theasterisks (* p<0.05; ** p<0.01; *** p<0.001). The data obtained from thestudy (FIG. 7) show that liposomes displaying ^(BPA)NeuGc andnitrophenyl (NP) induces tolerance to NP in mice.

The invention thus has been disclosed broadly and illustrated inreference to representative embodiments described above. It isunderstood that various modifications can be made to the presentinvention without departing from the spirit and scope thereof. It isfurther noted that all publications, patents and patent applicationscited herein are hereby expressly incorporated by reference in theirentirety and for all purposes as if each is individually so denoted.Definitions that are contained in text incorporated by reference areexcluded to the extent that they contradict definitions in thisdisclosure.

What is claimed is:
 1. A method for reducing antigen-specific antibodiesto a polypeptide antigen in a subject, comprising administering to thesubject a pharmaceutical composition comprising a liposome; a CD22,Siglec G, or Siglec-10 glycan ligand; and an effective amount of a Tcell-dependent MHC class II, B cell-specific polypeptide antigen that islinked to a lipid and co-displayed on the liposome with the CD22, SiglecG, or Siglec-10 glycan ligand; to thereby reduce production ofantibodies to the antigen by B cells in the subject.
 2. The method ofclaim 1, wherein the antigen is an autoantigenan or an alloantigen. 3.The method of claim 1, wherein the subject is a human.
 4. The method ofclaim 3, wherein the Siglec ligand is9-N-biphenylcarboxyl-NeuAcα2-6Galβ1-4GlcNAc (6′-BPCNeuAc),NeuAcα2-6Galβ1-4GlcNAc, or NeuAcα2-6Galβ1-4(6-sulfo)GlcNAc.
 5. Themethod of claim 1, wherein the polypeptide antigen is a self-antigen, anautoantigen, an alloantigen, a food antigen, or a tissue specificantigen.
 6. The method of claim 1, wherein the polypeptide antigen is aforeign antigen.
 7. The method of claim 1, wherein the administering isparenteral administration.